Aims: To increase cardiac output, β-adrenergic stimulation increases the rate and amplitude of cytosolic Ca²⁺ transients, elevating ATP consumption. At the same time, Ca²⁺ is taken up into mitochondria and activates the Krebs cycle, generating NADH for ATP production at the respiratory chain and NADPH to prevent oxidative stress. Mitochondria take up Ca²⁺ primarily via the uniporter (MCU) and release it via the Na⁺/Ca²⁺ exchanger (NCLX). Since Ca²⁺ extrusion is much slower than influx, steady-state mitochondrial Ca²⁺ concentration ([Ca²⁺]_m) is critically governed by heart rate. However, since basal heart rate varies ~10-fold across species, from ~60/min in human to ~600/min in mice, we speculated that there should be species-dependent differences in the activities of mitochondrial Ca²⁺ uptake and efflux to maintain [Ca²⁺]_m in a similar physiological, allowing Krebs cycle stimulation but preventing fatal opening of the permeability transition pore (mPTP).

Methods and results: Mitochondria were isolated from hearts of mice or rabbit ventricular- and human left atrial myocardium and exposed to sequential Ca²⁺ pulses of 10 or 20 µM at low (0 or 2.5 mM) or high (10 or 12.5 mM) extramitochondrial Na⁺, and extramitochondrial Ca²⁺ was determined by Calcium-Green 5N. Each Ca²⁺ application induced a sharp increase in extramitochondrial Ca²⁺, which subsequently decreased exponentially, indicating mitochondrial Ca²⁺ uptake via the MCU (since this was inhibited by the MCU blocker Ru360). At low extramitochondrial Na⁺, Ca²⁺-uptake velocities were not different between species, indicating that MCU activities are comparable. However, the net Ca²⁺ accumulation in mitochondria before opening of the permeability transition pore (mPTP, sensitive to cyclosporine A) was more than twice as high in human than mice. In mouse cardiac mitochondria, net mitochondrial Ca²⁺ accumulation was substantially reduced about 4-fold when extramitochondrial [Na⁺] was elevated to 10 or 12.5 mM, while this had smaller effects in rabbit (~2-fold decrease), but no effect in human mitochondria. To determine the kinetics of mitochondrial Ca²⁺ ([Ca²⁺]_m) uptake and release in working cardiac myocytes, we employed a patch-clamp based approach with simultaneous determination of cytosolic and mitochondrial Ca²⁺ concentrations, using Indo-1 AM salt and rhod-2 AM. With this approach, beat-to-beat [Ca²⁺]_m transients were observed during each cytosolic Ca²⁺ transient in either species. While the time to peak was faster in human vs. mouse cardiac myocytes, the rate of [Ca²⁺]_m decay was substantially slower in mouse vs. human mitochondria (tau, 1227±202 ms in humans, n=10; vs. 56±5 ms in mice, n=4; p<0.01).  

Conclusion: Steady-state mitochondrial Ca²⁺ accumulation is governed by strongly varying NCLX activities in a species-dependent manner while rates of mitochondrial Ca²⁺ uptake via the MCU are comparable across species. Also, the tolerance to net uptake of Ca²⁺ is larger in humans than mice. These insights have important therapeutic implications to guide strategies how to affect mitochondrial Ca²⁺ handling in heart failure, where diminished mitochondrial Ca²⁺ accumulation contributes to oxidation and reactive oxygen species emission.